Magnesium nitrate

AnimalsNinety virgin adult female Wistar rats (six week old, and weighing 250-300 g) were used in the present study. Through the experiment, the animals were maintained at the animal house under controlled conditions (12 hr light and dark cycle, 22°C and 60% relative humidity) with laboratory chow and water provided ad libitum (32 ).
all procedures involving animals were performed in accordance with the Guideline for Care and Use of Laboratory Animals of Mashhad University of Medical Sciences, Mashhad, Iran.
Study design and experimental groupsFemale rats were mated with males of the same strain. The day on which spermatozoa were found in the vaginal smear was designated as gestational day 0 (GD0). Then, the pregnant rats were divided into 9 groups randomly (n=10 in each group) as follows:
1- lead-exposed(L)group; the animals were treated with 1500 ppm lead acetate in drinking water starting at GD0. The lead exposure regimen was chosen based on a previous study (33 (link)).
2- lead + ascorbic acid (L+AA) group; the animals were treated with 1500 ppm leaded-water and ascorbic acid (500 mg/kg) via intraperitoneal injection (IP) once a day (22 (link)).
3- lead + garlic juice (L+G) group; the animals were received leaded-water and fresh garlic juice (1ml /100g/body weight) by gavage once a day (21 (link)).
4- lead + ascorbic acid +garlic (L+AA+G) group; the animals were treated with leaded water and ascorbic acid (500 mg/kg) via intraperitoneal injection and fresh garlic juice (1ml /100g/body weight) by gavage once a day (21 (link)).
5- Ascorbic acid (AA) group; the animals were treated with ascorbic acid (500 mg/kg) via intraperitoneal injection once a day.
6- Garlic (G) group; the animal were treated with fresh garlic juice (1ml /100g/Body weight) once a day by gavage.
7- Ascorbic acid + garlic (AA+G) group; the animal were treated with 500 mg/kg via intraperitoneal injection and fresh garlic juice (1ml /100 g/body weight) by gavage once a day.
8- Sham (Sh) group; animals were treated with tap water plus 0.4ml/l normal hydrogen chloride (HCl) and 0.5 mg/l Glucose.
9- Normal (N) group; animals were administrated with tap-drinking water.
All the treatments were continued during pregnancy and lactation (postnatal day 21=P21). After P21, pups were kept in the treatment regimens until P50.
Preparation of leaded water For the preparation of 1500 ppm leaded water, 30 g lead acetate, 8cc normal HCl (to avoid lead precipitation) and 10g glucose (for favorite taste) were dissolved in 20 liters of tap water.
Source of garlicFresh garlic bulbs were collected from a natural habitat around Mashhad during June to August 2011, and identified by botanists in Ferdowsi University of Mashhad, Iran and a voucher number deposited (FUMH: 39493).
Preparation of garlic juice To prepare garlic juice, garlic bulbs were separated, peeled and washed with distilled water. After drying in a shed, the clean garlic bulbs were crushed with an electric grinder and the extract was decanted carefully through muslin cloth (21 (link)).
Blood lead level measurement At the end of the experiment, the young pups were deeply anesthetized with chloroform and blood sample was taken transcardinally. To measure lead level in whole blood samples, a Perkin-Elmer Model 3030 atomic absorption spectrophotometer with a Perkin-Elmer HGA (Heat Graphite Atomizer) 400 graphite furnace and hydride system MHS 10 was used together with HCL (Hallow Cathode Lamp) and EDL (Electrode Discharge Lamp) for metal measurement of even low levels. Blood was diluted 1:10 with Triton X-100, with the addition of a matrix modifier containing ammonium phosphate monobasic and magnesium nitrate. All specimens were run in batches which included standard methods for calibration (Table 1). BLL was measured in each animal group before and after interventions in mothers (rats) as well as their offspring at the end of the experiment (P50).
Histological methodAt the end of treatment, the young pups were deeply anesthetized and their brains were removed carefully, washed in normal saline and fixed in normalized fixative containing 10% formaldehyde in 0.01 M phosphate buffered saline (PBS) overnight at room temperature. After fixation, the specimens were dehydrated with an ascending ethanol series, cleared with xylen and embedded in paraffin. The brain tissue blocks were cut into 5μm transverse serial sections and stained with Toluidine blue (32 ).
Quantification of DNsThe DNs were identified microscopically by cytoplasmic and nuclear condensation, shrinkage and hyperbasophilia properties in the hippocampal pyramidal cells and dentate gyrus granular cells.
The sections were scanned and photographed using a light microscope with a ×40 objective lens (UPlan FI, Japan), images transferred to computer using a high-resolution camera (BX51, Japan).
Morphmeterical methods were used to count DNs per unit area in CA1, CA3 and DG subdivisions of the hippocampus. The number of DNs was counted using a 10000 μm2 counting frame. The mean number of DNs per unit area (NA) in different regions of hippocampus was calculated using the following formula (33):
NA=∑Q̅a f . ∑PIn this formula “∑Q“ is the sum of counted particles appeared in sections, “a/f” is the area associated with each frame, and “∑P” is the sum of frame associated points hitting space.
Statistical analysisThe acquired data from BLL measurements and the DNs counting methods were reported as mean ± SE. For comparison of the lead blood level data obtained from before and after interventions in each group, paired sample t-test was used. The data resulted from pretreatment in each group of mothers was compared among all groups. The mean of lead blood level obtained at the end of experiments (after interventions) in mothers as well as their offspring at P50 in each group was compared in all groups. In addition, the mean number of DNs per unit area in each region of rat offspring hippocampus was compared in all groups.
To compare the lead blood level and the number of DNs per unit area among all groups, at first, data normality was assessed by the Kolmogorov–Smirnov test and then one-way analysis of variance (ANOVA) was used followed by Tukey post hoc test using SPSS software version 11.5. P≤0.05 was considered as significant.

Water-Vapor Transmission Rate (WVTR) was estimated according to the ASTM E96/E 96M-05 method. A handmade apparatus was used for such measurements of CS/PVOH/CuMt and CS/PVOH/TiMt films. Experiments were carried out at 38 °C and 50% RH. The methodology was described extensively in the literature [31 (link),32 (link),33 (link),34 (link),35 (link),36 (link),37 (link)]. The 2.5 cm diameter and 0.09 mm average thickness film was placed on the top of a one-open end cylindrical tube made of plexiglass which contained dried silica gel inside and was sealed by a rubber O-ring. The test tube was placed in a glass desiccator which contained 200 mL saturated magnesium nitrate solution (50% relative humidity (RH) at 38 °C). Test tubes were weighed periodically for 24 h and the WVTR [g/(cm²∙s)] was calculated according to the following equation: $$WVTR=\frac{\Delta G}{t·A}$$
where: ΔG (g) is the increase of weight of the tested tubes, t (s) is the time pass, ΔG/t (g/s) is the water transmission rate through the film which is calculated by the slope of the linear function ΔG = f(t), and A (cm²) is the permeation area of the film. Additionally, the weight of the tested films was measured before and after the WVTR test to exclude any absorption phenomena of humidity by the film.
For diffusion process through a membrane, Fick law [38 ] calculates the specific mass flow rate via the following equation: $$\frac{J}{A}=D\frac{\Delta C}{\Delta x}$$
where J (g/s) is the mass flow rate of a component through the membrane, A (cm²) is the membrane cross-sectional area permeated by this component, ΔC (g/cm³) is the concentration gradient of this component in the two sides of the membrane, and Δx (cm) is the membrane thickness.
Assuming that in our apparatus silica gel on one side absorbs the permeated water vapor totally and given that according to the ASTM E96/E 96M-05 method the humidity concentration in the opposite side of the film is 22.86747 g/cm³ (50% RH at 38 °C), then ΔC = 22.86747 g/cm³. For WVTR = J/A and combine Equations (2) and (3) we can calculate the diffusion coefficient D (cm²/s) for every film as follows: $$D_{WV}=WVTR·\frac{\Delta x}{\Delta C}$$
where WVTR [g/(cm²∙s)] is the water-vapor transmission rate, Δx (cm) is the film thickness, and ΔC (g/cm³) is the humidity concentration gradient in the two opposite sides of the film.

Experimental Design: The overall objective of the study was to uncover the mechanism by which non‐thermal plasma elicits immunogenic cancer cell death by evaluating the role of RONS generated. This was accomplished by 1) determining an ICD‐inducing regime of plasma, 2) identifying the RONS generated in that regime, and 3) delineating their effect by comparing direct DBD plasma treatment and treatment with exogenously prepared RONS solutions. Our initial screenings of RONS effect on ICD were performed in vitro on two melanoma cell lines and validated in an in vivo vaccination assay using syngeneic mice.
Cell Lines: The B16F10 murine melanoma cell line and the A375 human melanoma cell line were purchased from the American Type Culture Collection. Both cell lines were cultured in complete Dulbecco's modified Eagle medium containing 10% fetal bovine serum, 100 U mL⁻¹ penicillin, 100 µL streptomycin, and 4 × 10⁻³m l‐glutamine. Cells were cultured in a humidified environment at 37 °C with 5% CO₂.
Microsecond‐Pulsed DBD Plasma Parameters for Direct Treatment: A microsecond‐pulsed power supply was purchased from Advanced Plasma Solutions, and a 1.25 cm diameter copper DBD electrode was used for treatment in 24‐well plates. The copper electrode was covered with a 0.5 mm fused‐silica dielectric (Technical Glass) to prevent current arching. The microsecond‐pulsed power supply generated 17 kV pulses with ≈5 µs rise times and ≈1.5 µs pulse widths. The duty cycle was fixed at 100%.
Both cell lines were seeded into 24‐well plates at 3 × 10⁵ cells mL⁻¹ (0.5 mL per well) 1 d prior to plasma treatment. On the day of plasma treatment, medium was removed and cells were washed twice with PBS to remove serum and other organics from cell culture medium. PBS from the second wash was left in the well until right before plasma treatment. For DBD plasma treatment, PBS was removed and the DBD electrode was lowered into the well and positioned 1 mm above the cells with a z‐positioner (Figure 2A,B). Plasma was then discharged directly on the cells for 10 s at various pulse frequencies (50, 100, 250, and 500 Hz). Following plasma treatment, 500 µL of fresh cell culture medium was immediately added back into the well. Cells were incubated at 37 °C with 5% CO₂ for 24 h until further analysis. Mitoxantrone dihydrochloride (Sigma‐Aldrich, ≥97%, M6545), a chemotherapeutic used as a positive control, was diluted to a 5 mg mL⁻¹ stock solution and prepared into a working solution of 2 µg mL⁻¹ in complete medium. Cells were incubated with mitoxantrone for 24 h before collection and analysis.
Treatment with Pulsed Electric Fields: On the day of treatment, cells were washed with PBS and 1 mL of PBS or RONS solution (700 × 10⁻⁶m of H₂O₂, 1770 × 10⁻⁶m of NO₂⁻, and 35 × 10⁻⁶m of ONOO⁻) was added to each well immediately before treatment. The DBD electrode was then submerged into the liquid and operated at 17 kV and 500 Hz for 10 s, 1 mm above the cells. This method was to subject the cells to PEF generated from the microsecond‐pulsed power supply and DBD electrode without the production of plasma (Figure 2C). Following 10 s treatment, all the liquid was removed from the well and fresh cell culture medium was added. Cells were then incubated at 37 °C with 5% CO₂ for 24 h until further analysis.
DBD Plasma Treatment of Liquid: For liquid analysis, 50 µL of liquid (PBS or deionized water) was added into a 24‐well plate and distributed evenly across the bottom. The DBD electrode was positioned 1 mm above the surface of the liquid using the z‐positioner (Figure 2D). DBD plasma was generated at fixed voltage (17 kV), while the pulse frequency and treatment time were varied.
For preparation of plasma‐treated PBS for treatment of cells, PBS was treated for 100 s at 500 Hz. Prior to finishing the 100 s treatment, PBS was removed from the wells containing cells. All 50 µL of plasma‐treated PBS was then collected and added onto cells and 45 µL was removed. After 10 s treatment with 5 µL of remaining plasma‐treated PBS, 500 µL of complete medium was added back into the well and the cells were incubated for 24 h until further analysis.
Preparation of RONS Solutions and Treatment: The solutions of H₂O₂, NO₂⁻, and NO₃⁻ were prepared from commercially available H₂O₂ (Sigma‐Aldrich, ≥30%, 95321), sodium nitrite (NaNO₂) (Sigma‐Aldrich, ≥97%, 237213), and potassium nitrate (KNO₃) (Sigma‐Aldrich, ≥99%, P8394) dissolved in PBS (without iron, calcium, and magnesium). ONOO⁻ was prepared from NaOONO (Cayman Chemicals, ≥90% solution in 0.3 m sodium hydroxide, 14042‐01‐4) dissolved in PBS. Four different RONS solutions were prepared1)

H₂O₂/NO₂⁻/NO₃⁻—H₂O₂: 700 × 10⁻⁶m; NO₃⁻: 410 × 10⁻⁶m; NO₂⁻: 1360 × 10⁻⁶m

2)

H₂O₂/NO₂⁻—H₂O₂: 700 × 10⁻⁶m; NO₂⁻: 1770 × 10⁻⁶m

3)

ONOO⁻—35 × 10⁻⁶m

4)

H₂O₂/NO₂⁻/ONOO⁻—H₂O₂: 700 × 10⁻⁶m; NO₂⁻: 1770 × 10⁻⁶m; ONOO⁻: 35 × 10⁻⁶m

On the day of treatment, the cells were washed twice with PBS, to follow identical handling procedures with plasma treatment. PBS from the second wash was removed immediately before treatment and 50 µL of RONS solution was added into the well and rocked to ensure even distribution on cells. 45 µL was removed and the remaining 5 µL was left on the cells for 10 s. Following treatment, 500 µL of fresh, complete medium was added to the well and cells were incubated at 37 °C with 5% CO₂ for 24 h until further analysis. This procedure most closely mimics the process of direct DBD plasma treatment and is most realistic to the concentration of RONS generated by plasma and experienced by the cells.
Cell Survival Assay: Cell survival was quantified with a trypan blue exclusion test. Following 24 h incubation, cell supernatant was collected. Cells were then washed with 0.5 mL of PBS and detached with 200 µL of accutase. PBS from the wash was also collected with the cell supernatant. The cell suspension was collected, pooled with their supernatant and PBS wash, and homogenized by pipetting. A 50 µL sample was acquired and equal parts 0.4% trypan blue (Gibco, 15250‐061) was added to the sample. Cell counts were performed using a TC20 Automated Cell Counter (Bio‐Rad). The live cell concentration was recorded, and data were represented as a normalization to control.
CRT Expression from Cell Lines: CRT was measured using dual staining of PI and a monoclonal CRT antibody. Following 24 h after incubation, cells were washed with PBS, detached with 200 µL of accutase, and washed twice with 2 mL of FACS buffer (500 mL sheath fluid (BD Biosciences, 342003) + 2 g bovine serum albumin (Sigma, A9418) + 1 g NaN₃ (Merck, 1.06688.0100) in 100 mL H₂O). Each sample was split into two vials and one was stained with monoclonal primary rabbit anti‐CRT antibody (Abcam, ab196158) while the other was stained with rabbit IgG, monoclonal isotype control (Abcam, ab199091) for 40 min at 4 °C. Cells were then washed once with FACS buffer. 0.5 µL of PI was added to each sample immediately before being quantified with a flow cytometer. Fifteen thousand events were collected and only the PI− cells were analyzed for surface CRT expression. Data were analyzed and gated using the FlowJo software (FlowJo LLC, version 10). Data were expressed as percent CRT positive after accounting for nonspecific binding with their corresponding isotype. The gating strategy is described in detail in Figure S9 in the Supporting Information.
Mice and Antitumor Vaccination Assay: Thirty‐two 8‐week‐old female C57BL/6J mice were purchased from Charles River and housed in a pathogen‐free room at the Animal Center of the University of Antwerp. The sample size of this study (eight mice per group) was chosen using information in the literature7 and running an a priori power analysis using G*Power software (version 3.0.10). Input parameters included effect size (large, 0.8), α error probability (0.05), power (0.8), and number of groups (4). A total sample size of 24 was calculated to give an actual power of 0.859. Eight mice were randomly assigned to one of four groups, and housed during the whole of the experiment in four separate cages. Two mice from each group were housed in each cage. Investigators were not blind to the groups.
The vaccines for this assay were prepared from B16F10 melanoma cells exposed to 1) DBD plasma (500 Hz), 2) PEF + RONS (RONS: 700 × 10⁻⁶m of H₂O₂, 1770 × 10⁻⁶m of NO₂⁻, and 35 × 10⁻⁶m of ONOO⁻), or 3) mitoxantrone (2 µg mL⁻¹) in vitro, while untreated cells were used as a negative control. After treatment, cells were collected, washed twice with PBS, and resuspended in PBS at 10⁶ cells mL⁻¹. Cell suspension was incubated for 24 h at 37 °C with 5% CO₂ to reduce the viability of the cells and prevent subsequent tumor growth at the vaccination site.
On the day of vaccination, mice were shaved with electric clippers (to help visualize tumors) and injected with vaccine (10⁵ cells in ≈100 µL) on the right dorsal side. After 7 d, each mouse was injected with 10⁴ live B16F10 cells (in ≈100 µL) on the left dorsal side. Tumor size and growth were followed up to day 50 as defined prior to the start of the experiment. Three orthogonal diameters were measured using a digital caliper, and volumes were calculated using (4/3π)r₁ × r₂ × r₃. The humane study endpoint was set to when the total tumor volume exceeded 1500 mm³ or if tumors began to ulcerate. All animal experiments were approved by the University of Antwerp Animal Research Ethical Committee (ECD‐dossier 2017‐53).
Detection of H₂O₂: The H₂O₂ concentration was detected using potassium oxotitanate dehydrate (Alfa Aesar, 89620) solution in H₂O and H₂SO₄ (Sigma‐Aldrich, 95–98%, 258105M). Concentration of H₂O₂ in plasma‐treated samples was determined by UV–vis measurements performed on a Genesys 6 (Thermo Fischer) spectrophotometer with quartz cuvettes (10 mm light path, 2 mm internal width). Titanium(IV) reagent (0.1 m Ti, 5 m H₂SO₄) was prepared by dissolving 0.354 g of potassium bis(oxalato)oxotitanate(IV) dihydrate in a mixture of 2.72 mL of sulfuric acid and diluted to 10 mL with Milli‐Q water. 50 µL of plasma‐treated sample was added to the cuvette and diluted with 150 µL of PBS. 50 µL of sodium azide (NaN₃) (Sigma‐Aldrich, ≥99.5%, S2002) was added to the cuvette and thoroughly mixed. Afterward, 50 µL of Ti sulfate solution was added and homogenized. Air bubbles in the cuvette were eliminated with a sonicator (Branson 3200 ultrasonic bath) and water droplets were wiped from the cuvette before reading at 400 nm.
Detection of NO₂⁻ and NO₃⁻: A nitrate/nitrite colorimetric assay kit (Cayman Chemical, 780001) was used according to the provided protocol. To detect NO₂⁻ only, 50 µL of Griess reagent 1 (sulfanilamide) was added to each sample in a 96‐well plate, and 50 µL of Griess reagent 2 (N‐(1‐naphthyl)ethylenediamine) was immediately added afterward. The absorbance wavelength was read with a microplate reader Infinite 200 Pro (Tecan) at 540 nm. To detect NO₃⁻ and NO₂⁻, a nitrate reductase mixture (Cayman Chemical, 780010) and an enzyme cofactor mixture (Cayman Chemical, 780012) were added to each sample prior to the addition of Griess reagents. This allowed for the conversion of nitrate into nitrite. The absorbance was measured in duplicates and the samples were prepared in triplicates. The concentrations were calculated based on the obtained calibration curve.
EPR Spectroscopy Analysis: 50 µL capillaries (Ringcaps) were used to collect plasma‐treated samples, and a MiniScope MS200 spectrometer (Magnettech) was used to perform the analysis. After each plasma exposure experiment, the samples were immediately placed into a capillary tube. The overall time between exposure and analysis was 1 min. The general EPR parameters were as follows: frequency 9.4 GHz, power 3.16 mW (31.6 mW in case of (MGD)₂Fe₂⁺–NO), modulation frequency 100 kHz, modulation amplitude 0.1 mT, sweep time 30 s, time constant 0.1, and sweep width 15 mT. The simulated spectrum was double integrated to determine the concentrations reported here. Simulations were performed using hyperfine values obtained from the literature in the Spin Trab Database (National Institute of Environmental Health Sciences, 2018). EPR calibration was performed using solutions of 4‐hydroxy‐TEMPO (Sigma‐Aldrich, 97%, 176141) as reported elsewhere.33 Specific spin traps and other molecules were used to detect RONS in the liquid (Table2). All recorded experimental EPR spectra and simulations are shown in Figure S1 in the Supporting Information, along with the corresponding hyperfine values used. All fixed‐pulsed experiments presented in the results were performed with three to five replicates unless otherwise specified.
Detection of O/¹O₂/O₃: TEMP spin trap was dissolved in PBS (50 × 10⁻³m) to detect O/¹O₂/O₃ following DBD plasma treatment (Sigma‐Aldrich, ≥99%, 115754). TEMP reacts with these oxygen species to form the spin adduct TEMPO, which can be detected by means of EPR spectrometry. To determine the contribution of O/O₃, 100 × 10⁻³m sodium azide (NaN₃) (Sigma‐Aldrich, ≥99.5%, S2002) was added to the TEMP solution before plasma treatment to quench ¹O₂. Therefore, the collected spectrum of TEMPO was a result of the remaining oxygen species.
Detection of ^•OH and O₂^•− with EPR Spectroscopy: DEPMPO spin trap (Enzo Life Sciences, ≥99%, ALX‐430‐093) was dissolved in PBS (100 × 10⁻³m) to detect ^•OH and O₂^•−. DEPMPO reacts with O₂^•− to produce DEPMPO–OOH while it reacts with ^•OH to produce DEPMPO–OH. Experiments using 50 Hz pulse frequency were performed once with treatment times of 10 s or more.
Detection of ^•NO with EPR Spectroscopy: The spin probe PTIO (Enzo Life Sciences, ≥98%, ALX‐430‐007) was dissolved in PBS (200 × 10⁻⁶m) to detect ^•NO from DBD plasma treatment. ^•NO reacts with PTIO to form PTI that can be detected through EPR spectroscopy. When pulse frequency was varied from 50 to 500 Hz, treatment time was fixed at 50 s in order to generate detectable levels of ^•NO.
The MGD spin trap (Enzo Life Sciences, ≥98%, ALX‐400‐014) was also used to detect ^•NO. MGD was dissolved in deionized water (20 × 10⁻³m) and combined with Fe(II)SO₄·7H₂O (4 × 10⁻³m) (Sigma‐Aldrich, ≥99%, 215422). This solution was treated with DBD plasma and Na₂S₂O₃ (100 × 10⁻³m in deionized water degassed with argon) (Sigma‐Aldrich, ≥98%, 72049) was immediately added to the sample and collected for EPR analysis. When pulse frequency was varied from 50 to 500 Hz, treatment time was fixed at 120 s in order to generate detectable levels of ^•NO.
Detection of ONOO⁻with LC–MS: Solutions of 100 × 10⁻⁶m l‐tyrosine (Sigma‐Aldrich, ≥98%, T‐3754) and 100 × 10⁻⁶m diethylenetriaminepentaacetic acid (Sigma‐Aldrich, ≥98%, D1133) in 2 × PBS were exposed to plasma for a given period of time, as described by Wende et al.31 The solutions were collected and flash frozen immediately after exposure.
The separation and detection of 3‐nitrotyrosine was done by a Waters ACQUITY ultraperformance liquid chromatograph (UPLC) coupled to a Waters triple quadrupole mass spectrometer (Xevo TQ MS). The used column was a Waters ACQUITY UPLC HSS T3 2.1 mm × 100 mm column (1.8 µm particles), heated to 40 °C. The 9 min gradient was used for separation with A) water containing 0.1% formic acid and B) acetonitrile containing 01% formic acid, at a flow rate of 0.6 mL min⁻¹: 0–1.0 min 2% B, 1.0–4.0 min 2% to 18% B, 4.0–5.0 min 18% to 97% B, 5.0–6.0 min 97% B, 6.0–7.0 min 97% to 2% B, and 7.0–9.0 min 2% B. The parameters used in electrospray ionization tandem mass spectrometry in positive mode were as follows: capillary, 0.5 kV; cone, 22 V; extractor, 3 V; source temperature, 150 °C; desolvation temperature, 600 °C; desolvation gas flow, 1000 L h⁻¹; cone gas flow, 0 L h⁻¹; collision gas flow, 0.15 mL min⁻¹; collision energy, 2 V.
A multiple reaction monitoring (MRM) method application was optimized for the detection of tyrosine (transition m/z 182–136) and 3‐nitrotyrosine (transition m/z 227–181). For calibration of 3‐nitrotyrosine, eight standard solutions were made ranging from 0 to 10 × 10⁻⁶m and analyzed using the MRM method. The samples were diluted to a starting concentration of 10 × 10⁻⁶m tyrosine in 95% water and 5% acetonitrile containing 0.1% formic acid.
Statistical Analysis: Statistical differences for cell survival and CRT expression were analyzed using the linear mixed model with JMP Pro 13 (SAS software). The fixed effect was the treatment, and the random effects included were the different dates the experiment was performed and the flasks the cells used were split from. The interactions between the treatment and the date as well as interactions between the treatment and the flasks were tested. The random slope model was used when the interactions were significant (P < 0.05) and the random intercept model was used in all other cases. The fixed effect tests determine whether there was a significant difference between treatments (P < 0.05). When the difference is significant, the Dunnett's test for statistical significance was used to calculate adjusted P value compared to the control. A P value of <0.05 was considered statistically significant. For all in vitro experiments, treatment conditions were performed in duplicates on the same day and repeated on three separate days as a minimum. The total number of observations for each treatment group is defined in the figure or figure legend. The survival curve of the vaccination study was prepared in Graphpad Prism and compared using the log‐rank (Mantel–Cox) test. A P value of <0.05 was considered statistically significant. All figures were prepared in Graphpad Prism (Graphpad Software). For all chemical species, a nonlinear regression was used to determine the best‐fit line and R² value with a y‐intercept constraint at zero. Analysis was performed and figures were prepared in Graphpad Prism (Graphpad Software). No data were excluded.

The reagents used in this study, including hydrogen peroxide (H₂O₂), nitric acid (HNO₃), and sodium nitrate (NaNO₃), were purchased from the Guangzhou Chemical Reagent Factory. Magnesium chloride hexahydrate (MgCl₂·6H₂O), cadmium nitrate tetrahydrate (Cd(NO₃)₂·4H₂O), and lead nitrate (Pb(NO₃)₂) were purchased from Macklin Reagent Company.

Fresh Mentha pulegium plant was collected from Narm village in South Khorasan, Iran. Magnesium nitrate hexahydrate (Mg (NO₃)₂·6H₂O), Zinc nitrate hexahydrate (Zn (NO₃)₂·6H₂O), Silver nitrate (Ag (NO₃)₂), Methylene blue (C₁₆H₁₈ClN₃S), Rhodamine B (C₂₈H₃₁ClN₂O₃), and Sodium hydroxide (NaOH) were purchased from Merck and Sigma Companies.